Clear cell renal cell carcinoma (ccRCC) is the most common cancer arising in the kidney, and is a highly aggressive and lethal disease in its metastatic form. The phenotype of this cancer is dominated by two features: inherent resistance to chemotherapy and excessive vascularization of tumors. These tumors are intensely angiogenic as a result of loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene, which allows the constitutive stabilization of the hypoxia inducible factors (HIF-1 and HIF-2), creating a pseudohypoxic environment inducing numerous factors promoting endothelial growth. As a result of these biological discoveries, treatments for ccRCC have focused on VEGF pathway targeting. However, acquired resistance to VEGF pathway targeted agents occurs universally, via vascular reprogramming signals that circumvent VEGF signals to restore tumor angiogenesis. One signal mediating this resistance is the Notch pathway, which is stimulated by the ligand DLL4 and promotes growth of vessel sprouts and coordinates effective endothelial vessel formation. In preliminary studies, inhibition of DLL4 by a neutralizing antibody reduced the growth of ccRCC xenografts and produced disruption of the normally robust vasculature of these tumors. We hypothesize that ccRCC is highly responsive to Notch signals as a result of the pseudohypoxic phenotype, and that VHL mutant ccRCC will be exquisitely sensitive to DLL4 inhibition. We further hypothesize that the vascular phenotype of these tumors can be used to monitor the potential for sensitivity to anti- angiogenic therapies, as well as to characterize the patterns of disease resistance resulting from angiogenic reprogramming. To simultaneously measure the vascular architecture, tumor perfusion, and tumor vessel molecular characteristics, we have turned to contrast enhanced ultrasound with molecular imaging of endothelial VEGFR2 as novel tool for making in vivo measurements of critical tumor elements at key transition points in treatment. Finally, based on proposed activity of DLL4 inhibitor to increase VEGFR2 expression and promote sprouting, we will test the hypothesis that DLL4 inhibition might be most effective following emergent resistance to treatment with a VEGFR2 tyrosine kinase inhibitor. DLL4 Ab may provide an effective second line agent in this setting, or by producing VEGFR2 upregulation on endothelial cells, decreasing pericyte coverage, and disorganized sprouting, restore tumor vessels to sensitivity to anti-VEGFR2 agents. The successful completion of these aims will advance our understanding of Notch signaling in RCC vascular biology, and will advance a promising agent for future use in clinical trials for treatmen of resistant disease.